Sequestosome 1/p62 (p62) is a scaffold/adaptor protein with multiple functions implicated for neuronal and bone diseases. It carries a ubiquitin binding domain through which it mediates proteasome-dependent proteolysis. In addition, p62 is reported to regulate NF- B activity in some cells. To date, however, the role of p62 in innate immunity has not been fully elucidated. In this study, we found that IFN- plus TLR signaling stimulates late expression of p62 in murine M&#61510;. Overexpression of p62 inhibited expression of multiple cytokines, IL-12p40, TNF- , IL-1&#946;, IL-6, and IFN-&#946;, whereas p62 underexpression by small hairpin RNA markedly elevated their expression, indicating that p62 is a broad negative regulator of cytokine expression in stimulated M&#61510;. We have shown that p62 interacts with IRF8 and Ro52, the transcription factor and ubiquitin E3 ligase that are important for IL-12p40 expression. This interaction, detectable at a late stage in stimulated M&#61510;, led to increased polyubiquitination and destabilization of IRF8. Upon M&#61510;stimulation, p62 bound to TNFR-associated factor 6, another E3 ligase important for NF- B activation, but later this interaction was replaced by the recruitment of the deubiquitinating enzyme, cylindromatosis, an inhibitor of NF- B activity. Recruitment of cylindromatosis coincided with reduced TNFR-associated factor 6 autoubiquitination and lower NF- B activation. Our results indicate that p62 orchestrates orderly regulation of ubiquitin modification processes in M&#61510;to ensure attenuation of cytokine transcription postactivation. Together, p62 may provide a mechanism by which to control excessive inflammatory responses after M&#61510;activation. Previously we have shown that IRF3 and IRF7, as well as IRF8 are conjugated to a small ubiquitin like peptides SUMO in response to toll like receptor (TLR) and retinoic acid like receptor (RLR) signaling. This is catalyzed by However, a SUMO E3 ligase that catalyzes covalent SUNO conjugation to the IRF proteins has not been identified. Toward this end we have begun analyzing the role of Piasy in ype I IFN transcription. PIASy is a member of the PIAS family that includes four SUMO ligases. We found that PIASy among other family members is a likely candidate involved in regulating type I IFN transcription, because when ectopically expressed Piasy repressed IRF7 mediated IFN promoter activity most strongly relative to other PIAS members. In line with this result, Piasy-/- embryonic fibloblasts (MEFs) induced type I IFNs at much higher levels than wild type cells, when stimulated by viruses, TLR and RLR signals. Furthermore IRF3 and IRF7 were SUMOylated by both PIAS1 and PIASy. PIASy and other PIAS members have several domains including a protein-protein interaction domain, the catalytic domain containing a zinc finger motif and SUMO interaction motif. Our results show that while the N-terminal domain is not required, the central SUMO catalytic domain and the C-terminal SUMO binding motif play important role in regulating IFN transcription.